1. Field of the Invention
The present invention relates to recombinant DNA cloning vectors which are useful for identifying eukaryotic DNA regulatory sequences. More particularly, the present invention relates to cloning vectors which contain an origin of replication that allows replication in a eukaryotic cell, a gene coding for an activator of the origin, and a DNA segment which provides a means for detecting the increased replication of the vector. A cloning site is provided upstream of the gene for cloning eukaryotic DNA sequences.
2. Description of the Prior Art
Gene expression in prokaryotes and eukaryotes can be regulated in many ways, including control of transcription, control of post-transcriptional processing, and control of translation. Gene expression can be regulated first at the developmental level, secondly at the tissue level, the thirdly at the cellular level. At the developmental level, the gene can be regulated so that it is expressed only during a specific developmental stage. The control at the tissue level occurs so that a gene will be expressed only in the proper tissue. Regulation at the cellular level occurs when the gene is being expressed at the correct developmental stage and in the correct tissue to control the amount of gene product produced. For example, the .gamma. chain of hemoglobin is expressed in the human fetus, but is not expressed in the normal human adult. Thus there is a developmental control on the time of production of this protein. During this time of development, the expression of the gene for the .gamma. chain is regulated so that it occurs in the erythocyte precursor and can further be regulated by other mechanisms including those listed above.
The identification of eukaryotic regulatory sequences has many uses. The sequences which have been identified can be used in eukaryotic expression vectors. The sequences can be inserted into an appropriate site in the expression vector to regulate the expression of the desired gene. If the regulatory sequence is a strong promoter, it will drive the transcription of the desired gene to a greater extent than the natural promoter. If the regulatory sequence is an enhancer, it will enhance the transcription of the desired gene. Some regulatory sequences may be controlled by a modulator. The presence or absence of the modulator can increase or decrease the transcription of the desired gene. Thus, by appropriate control of the regulatory sequence, the gene expression can be increased. Use of regulatory sequences controlling gene expression at the tissue level is important, when the gene is inserted into tissue which may be lacking the gene (for example, as the result of a genetic disorder). Eukaryotic regulatory sequences which have been utilized for expression of genes in eukaryotic expression vectors include yeast promoters, such as alcohol dehydrogenase I, viral promoters such as the SV40 T antigen promoter, and enhancers such as those from SV40. Eukaryotic regulatory sequences identified by the present vectors can be substituted for prior art eukaryotic regulatory sequences to provide new eukaryotic expression vectors. In addition, a desired gene can be inserted into the present vectors containing a known eukaryotic regulatory sequence to produce amplification of the gene, resulting in increased expression.
Vectors have been prepared which control gene amplification as a result of increasing the replication of the vector. One such example is described in Larsen et al, Gene 28, 45 (1984). The vector described therein is a bacterial vector and contains a bacterial origin of replication, a bacterial gene which produces a product to activate the origin of replication, and a marker. A regulated promoter is placed upstream of the gene which can lead to increased production of the product to stimulate vector replication. These vectors have been termed conditional runaway replication vectors.
The present invention provides a vector for identifying eukaryotic regulatory sequences efficiently and quickly. The vector utilizes a gene for detection (detection gene) which can be identified quickly without the need for growing colonies. Since the vector amplifies the detection gene through increased replication, the efficiency of detection increases significantly.